Manufacturing process for porous material

ABSTRACT

A method for the manufacture of a solid porous separation material based on a polysaccharide, said method comprising the steps of: (a) providing an aqueous solution (I) of a polysaccharide, (b) solidifying the solution, preferably by transforming the solution to a gel, and (c) optionally cross-linking the polysaccharide, with the proviso that, if step (c) is present, steps (b) and (c) may be carried out simultaneously. The method is characterised in that the polysaccharide provided in step (a) is modified by being inter-molecularly cross-linked to an extent such that the viscosity of solution (I) is at least 110%, preferably at least 200%, of the viscosity of an aqueous solution (II) of the corresponding polysaccharide which has not been inter-molecularly cross-linked and which is present in the same concentration as the inter-molecularly cross-linked polysaccharide is in solution (I).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/451,194 filed Jun. 19, 2003, which is a filingunder 35 U.S.C. § 371 and claims priority to international patentapplication number PCT/EP2001/015014 filed Dec. 19, 2001, published onJul. 11, 2002 as WO2002/053598 and also claims priority to Swedishpatent application number 0004928-8 filed Dec. 29, 2000; the disclosuresof which are incorporated herein by reference in their entireties.

TECHNICAL FIELD AND BACKGROUND TECHNOLOGY

The present invention is a method for the manufacture of a porousmaterial from a polysaccharide. The starting polysaccharide used in theinnovative method has been modified such that it will give an aqueoussolution (solution I) which has a viscosity that is higher than theviscosity of the corresponding solution (solution II) in which theunmodified polysaccharide is present in essentially the sameconcentration. Other conditions for solutions I and II are the same.

By the term “unmodified polysaccharide” is meant the polysaccharide orthe polysaccharide derivative that has been used to synthesise themodified polysaccharide that has an increased viscosity.

According to previous techniques, processes comprising the steps of:

-   -   (a) forming an aqueous solution (I) of a suitable        polysaccharide,    -   (b) solidifying the solution, and    -   (c) optionally cross-linking the polysaccharide to become        insoluble have resulted in porous polysaccharide material.        Steps (b) and (c) has been performed simultaneously or in        sequence. The term “solidifying” primarily has meant        transforming the solution to a gel.

The material so obtained has been used as support matrices in separationmethods, such as electrophoresis, chromatographic and batch-modeseparations based on adsorption and/or size exclusion, cell culturing(as microcarriers) etc.

This production technology can give beads if the solution is emulsifiedin an organic solvent, which is not miscible with water (water-in-oilemulsions). By including the proper derivatization reagent in thesolution, it will be possible to form inter- as well as intra-molecularcross-links to an extent that will insolubilize the polysaccharide andsolidify the drops. An insoluble cross-linked 3-dimensional polymericpolysaccharide network will form. An alternative way to produce beadshas been to select a polysaccharide that dissolves in aqueous liquidmedia when warmed and solidifies to a gel when the temperature of thesolution is decreased. In this variant the polysaccharide may becross-linked simultaneously or subsequent to the gelling reaction.

In the case the solution is transformed to a gel without prioremulsification monolithic material will form.

Cross-linking is imperative for gel formation in case the polysaccharideis of the kind that lacks or has a too low gelling temperature.Otherwise cross-linking is optional and depends on use.

Cross-linking means that the rigidity of the material will increasewhich in turn means that the material may be better fitted to usesrequiring application of pressure, for instance liquid chromatography.

The pore sizes of the material obtained primarily depend on theconcentration of polysaccharide in the solution provided in step (a).

Sometimes monolithic materials with very large pores (macropores orsuperpores) are desired. Typical large pores have diameters >0.2 μm,such as ≧0.5 μm. In this case one starts with forming an oil-in-watersolution where the water phase comprise the polysaccharide. The systemis then transformed to a gel by cooling and/or cross-linking in the samemanner as for a material with smaller pores. In the case beads withlarge pores are desired, the oil-in-water emulsion is emulsified into anorganic solvent that is not miscible with water, whereafter the aqueoussolution is transformed to a gel in the same manner as discussed above.See U.S. Pat. No. 5,723,601 (Amersham Pharmacia Biotech AB), WO 0017257(Amersham Pharmacia Biotech AB) and WO 0012618 (Amersham PharmaciaBiotech AB).

Alternative ways for producing porous polysaccharide beads includeso-called atomisation techniques in which free drops of thepolysaccharide solution (solution I) is created. These variants can beillustrated by spraying the solution in an air stream (WO 9702125, FMCCorporation) and WO 0029466, XC Corporation)) or by the so-calledspinning disc atomisation (WO 9520620 (Biodev AB)).

Alternative ways include coating of individual solid particles with thepolysaccharide solution (solution I) prepared in step (a) followed bysolidifying the solution as in step (b) with an optional subsequentcross-linking (step (c)). See for instance WO 9200799 (UpfrontChromatography AS).

WO 9738018 discloses a process for the production of a cross-linkedpolysaccharide gel. More specifically, this application teaches a methodof preactivating an agarose solution to a suitable degree. After such apreactivation, the solution is solidified and the activated group canthen be used as a cross-linker to improve the rigidity of the gel.

U.S. Pat. No. 5,827,937 also relates to providing an activatedpolysaccharide. This is achieved by sterically preventing completion ofthe cross-linking, whereby an activated polysaccharide is obtained. At alater stage, the polysaccharide is reactivated and the cross-linking isthen allowed to proceed to a viscoelastic gel.

DRAWBACKS OF EARLIER TECHNIQUES

The above-mentioned processes have certain drawbacks. For instance, ifone is to incorporate densifying particles by dispersing the particlesinto the solution, the viscosity of the solution is typically not highenough. The densifying particles will have strong tendency to separateout without being incorporated into the final polysaccharide material.In the case the goal is to produce polysaccharide beads containingdensifying particles the portion of beads without densifying materialwill be unacceptable high. This drawback in particular applies for thekind of beads disclosed in WO 971732 (Amersham Pharmacia Biotech AB)(bead density >1 g/cm³ such as ≧1.2 g/cm³ (in a wet swollen state) anddensifying particles with a density ≧3 g/cm³).

It is also likely that viscous compared to non-viscous polysaccharidesolutions have advantages in the so-called atomisation techniques.Compare e.g. WO 9520620 (Biodev AB).

These problems can not simply be overcome by conventionally increasingthe viscosity by (a) increasing the concentration of the polysaccharide,(b) cooling the solution, (c) adding thickeners etc. Increasing theconcentration of the polysaccharide leads to decreased pore sizes, whichis a drawback if material with smaller pores is to be produced. Coolingis of limited value due to the fact that most polysaccharides andpolysaccharide derivatives precipitates or gels when cooled. Addition ofthickeners tends to give inhomogeneities in the final material.

OBJECTIVES OF THE INVENTION

The objectives of the invention are to provide a manufacturing method asdefined above in which the above-mentioned drawbacks have beenminimised. This in particular applies to the manufacture of beadedmaterial containing one, two, three or more densifying particles beadincluding coating procedures and the manufacture of beads by atomisationtechniques such as spinning disc atomisation and spraying.

THE INVENTION

The invention thus is an improved variant of the method given in theintroductory part. The main characterising feature is that thepolysaccharide used is inter-molecularly cross-linked to an extent suchthat the viscosity of solution (I) is at least 110%, preferably at least200% such as at least 500%, of the viscosity of an aqueous solution (II)of the corresponding polysaccharide which has not been inter-molecularlycross-linked. The concentration of the corresponding polysaccharide insolution (II) is essentially the same as the concentration of theinter-molecularly cross-linked polysaccharide in solution (I).

Accordingly, the present invention provides a method wherein theviscosity of the polysaccharide is influenced or set before thesolidification and this can be performed without any effect on the poresize in the final product. Accordingly, the viscosity is set to apredetermined value, as compared e.g. to the above-discussed WO9738018wherein the viscosity will be a result of the preactivation, which isthe main aim. Thus, said WO 9738018 accepts the viscosity obtained foreach preactivation, contrary to the present invention, which is directedto obtaining a desired viscosity.

As discussed above the term “corresponding polysaccharide” refers to thepolysaccharide or polysaccharide derivative which has been derivatizedto an increased viscosity. Derivatisation in this particular contextthus includes any side reaction that takes place in parallel with theintroduction of inter-molecular cross-links. A typical side reaction isintra-molecular cross-linking.

This kind of derivatization can be accomplished by the aid of apolysaccharide chain association reagent, which typically acts as across-linking reagent. In other words this kind of reagents permitsformation of bridges between polysaccharide chains thereby making thechains longer and the corresponding solutions more viscous.

There are mainly two kinds of polysaccharide association reagents:

-   -   (1) bifunctional reagents (including multifunctional reagents)        in which each functional group is capable of reacting directly        with a hydroxy group of the polysaccharide or an activated form        thereof to give a covalent bond (homobifunctional reagents), and    -   (2) bifunctional reagents (including multifunctional reagents)        in which only one functional group is capable of forming a        covalent bond with the polysaccharide while the other functional        groups can be activated for reaction after the first function        has reacted (heterobifunctional reagents).

Directly reactive functional groups can be illustrated withelectrophilic groups such as epoxides; haloalkyl groups such ashalohydrins, vicinal dihalides, α-halocarbonyls; activated esters; acidhalides etc. In one and the same chain association reagent, the reactivefunctional groups may be identical or different.

Functional groups in the chain association reagent that requireactivation of the hydroxy group of the polysaccharide are typicallynucleophilic, such as amino, hydroxy etc. Activation of hydroxy groupsof the polysaccharides often means transformation to electrophilicgroups, for instance of the type given in the preceding paragraph.

Bifunctional reagents of the second type (2) are illustrated by reagentsin which

-   -   (a) the activatable function is an unsaturation, i.e. a        carbon-carbon double or triple bond, and    -   (b) the other function is represented by groups that are        directly reactive with hydroxy in the polysaccharide or an        activated form thereof. The directly reactive group of these        reagents can be selected according to the same rules as for the        type (1) reagents. Once inserted onto the polysaccharide the        groups with a concealed reactivity may be activated, e.g. by        halogenation or epoxidation. Alternatively they may be caused to        react with each other, for instance via free radical reactions.        Typical examples of popular unsaturated groups are alkene groups        such as in allyl and in acryl esters, acryl amides and the        corresponding methacryl variants.

The intra-molecularly cross-linked polysaccharide provided in step iswater-soluble. It is typically based on a native polysaccharide that iswater-soluble as such or that has been derivatized to becomewater-soluble. Derivatization in this context means that groups that arehydrophilic as such or that disturb the native conformation of awater-insoluble polysaccharide have been introduced. Accordingly theinter-molecularly cross-linked polysaccharide may be based on a nativepolysaccharide such as dextran, agarose, cellulose, starch, pullulanetc. For some kind of porous polysaccharide matrices it is an advantageto use a polysaccharide in step (a) that dissolves in warm water butgels or solidifies upon cooling. Typical examples of this kind ofpolysaccharides are agarose and certain modified forms thereof,hydroxyalkylated celluloses etc.

Derivatization of native polysaccharides that then can beintra-molecularly cross-linked and used in step (a) means introductionof organic groups such as charged groups (e.g. carboxy groups, sulphonicacid groups, ammonium groups), neutral groups that may be hydrophilic(e.g. hydroxy lower alkyl groups and lower alkyl groups) etc. By loweralkyl is typically meant C₁₋₃ alkyl groups (methyl, ethyl, propyl) orany other alkyl group that introduces the desired properties on thenative or derivatized polysaccharide.

The degree of cross-linking in the polysaccharide that is provided instep (a) should be such that the polysaccharide is water-soluble at thetemperature used for producing solution (I). This means that theconditions and reagents used for its synthesis should be selected suchthat unacceptable amounts of inter- and intra-molecular cross-links arenot formed.

A suitable quality of a polysaccharide for step (a) may be obtained byusing a very low amount of chain association reagent compared to theamount of polysaccharide. If the amount is too high the risk for quickinsolubilization becomes significant. The actual amount will depend onfactors such as molecular weight of the reagent, its reactive groups andtheir ability to participate in side reactions, the amount ofpolysaccharide etc. There are advantages in monitoring the proceeding ofthe reaction by the measurement of the change in viscosity and stoppingthe reaction when the desired viscosity has been obtained. See theexperimental part for a general guideline how to control the synthesis.

It has been found that the concentration of the inter-molecularlycross-linked polysaccharide required for predetermined porosity may beessentially the same as the concentration of the correspondingnon-cross-linked polysaccharide. This gives practical advantages sincethe concentrations of the polysaccharide that is provided in step (a)can be set according to well-established practice while at the same timeavoiding the drawbacks discussed above.

Porosity values and/or pore sizes of polysaccharide matrices are oftenexpressed as exclusion limits in terms of how large portion of thematerial a particular compound can utilise (Kav). See Hagel in “ProteinPurification, Principles, High Resolution, and Applications”, J-C Jansonand L Rydén (Eds), VCH Publishers Inc. New York, 1989, p. 99. TypicalKav values are within the interval of 0.10-0.95 and may depend also onfurther derivatization of the material, for instance introduction ofso-called extenders (WO 9822572, Amersham Pharmacia Biotech AB). Thismeans that for the matrix as it is obtained in step (c), the typicalinterval is somewhat narrower such as 0.40-0.95.

The polysaccharide material will always contain so-called smaller pores(micropores) in which mass transport is taking place by diffusion. Inaddition there may also be present larger pores (macropores orsuperpores) in which mass transport can take place by convection. Thesize range for the micropores is typically 20-5000 Å and for thesuperpores 0.5-100 μm. For material in form of porous beads, the ratiobetween the pore diameters of the macropores and the bead diametertypically is in the interval 0.01-0.3, with preference for 0.05-0.2. Theratio between the pore diameters of the micropores may in the preferredvariants extend up to 0.05 but is otherwise below 0.01.

See for instance WO 0017257 (Amersham Pharmacia Biotech AB), WO 0012618(Amersham Pharmacia Biotech AB) and WO 9319115 (Amersham PharmaciaBiotech AB).

The material can be in form of a population of porous beads or a porousmonolith.

The mean bead diameter may vary depending on the use but as a generalrule is within the interval of 1-1000 μm, preferably 1-50 μm for highperformance applications and 50-300 μm for preparative purposes. Apopulation of beads produced may be mono disperse (monosized) or polydispersed (polysized). By a mono disperse population of beads iscontemplated that more than 95% of the beads have diameters(hydrodynamic diameters) within the mean diameter of the population ±5%.The manufacture of beads having a certain mean bead diameter and beadsize distribution is done according to established practice.

Beads having densities above about 1 g/cm³ (in a wet swollen state) areused in separation methods involving adsorption to beads that have beenfluidised by an upward liquid flow. The liquid then typically is aqueouswith a density around 1 g/cm³. This kind of beads may be produced by theaid of the present invention which then means that densifying minorparticles are dispersed into the aqueous solution prepared in step (a)whereafter the solution is made into drops that are allowed to solidify.There may be one, two or more filler particles per bead. A producedpopulation of beads may have a distribution of densifying particles inthe beads or the same number of densifying particles in each bead. Thedensifying particles may be porous or non-porous. This kind of beadshave been described in WO 9218237 (Amersham Pharmacia Biotech AB); WO9717132 (Amersham Pharmacia Biotech AB); WO 9833572 (Amersham PharmaciaBiotech AB); and WO 9200799 (Kem-En-Tek/Upfront Chromatography A/S).This kind of beads may also be produced by so called atomisationtechniques that have been discussed in general terms above. Beads nothaving the desired density are removed, for instance beads lacking orhaving too many densifying particles.

According to the invention this kind of heavy polysaccharide beads mayalso be prepared by coating individual densifying particles by thepolysaccharide solution provided in step (a). After coating the solutionis allowed to solidify (step (b)). The densifying core particle may beporous in order to accomplish beads with large surface area. See U.S.Pat. No. 5,837,826 (Flickinger et al).

The densifying particles are in most cases based on inorganic materialfor instance glass, quartz, metal, metal alloy, metal salts etc. Typicaldensities of the particles are ≧1.5 g/cm³. See WO 9218237 (AmershamPharmacia Biotech AB) and WO 9717132 (Amersham Pharmacia Biotech AB).The latter explicitly discloses densifying material that is below andabove, respectively, 3 g/cm³ (but always above 1 g/cm³). The relativeamount of the densifying material may vary depending on the use of theend product, but typically it constitutes from a 1-5% up to about 70%based on volume per volume. For coated densifying particles the particlemay constitute up to 90-95% of the total bead volume.

The material may be derivatized to contain functional groups (ligands)that are used in an adsorption method to bind a desired substance to thematerial. Functionalization is typically taking place after step (c),but may if the ligand does not to any significant negative extentinterfere with steps (a) through (c) be done before step (a). Typicalligands are members of so called affinity pairs, more particularbio-affinity pairs.

Well-known affinity pairs (ligand-receptor pairs) are

-   -   (a) oppositely charged entities (ion exchange groups; the        immobilised entity being selected among primary, secondary,        tertiary and quaternary ammonium, sulphonate, sulphate,        phosphonate, phosphate, carboxy etc groups),    -   (b) antibodies and antigens/haptens,    -   (c) lectins and carbohydrate structures,    -   (d) IgG binding proteins and IgG (Protein A and IgG, Protein G        and IgG etc),    -   (e) pair of hydrophobic groups,    -   (f) chelators and chelates,    -   (g) complementary nucleic acids,    -   (h) cells and cell binding ligands,

Affinity members also include entities participating in catalyticreactions, for instance enzymes, enzyme substrates, cofactors,co-substrates etc. Members of cell-cell and cell-surface interactionsand a synthetic mimetic of bio-produced affinity members are alsoincluded.

A further kind of ligands is able to create reversible covalent bondsduring the adsorption step, for instance by containing so calledreactive disulphides, —S—SO_(n)— where n is an integer 1 or 2 and thefree bonds bind to saturated and/or unsaturated and/or aromatic carbons.

Step (b) above is carried out by any of the known techniques forpreparing porous monoliths or population of beads from polysaccharidesolutions. Thus monoliths may be obtained simple by cross-linking theintra-molecularly polysaccharide dissolved the solution (I), or, ifmacroporous materials are desired, by first forming an inverse emulsionfrom the solution.

Material in beaded form may be obtained by emulsifying the solution inan organic solvent not miscible with water before solidification of thesolution. In the alternative, atomisation techniques or conventionalcoating procedures of particulate material are applicable as well. Seeabove.

For more details of applicable techniques, see the publicationsdiscussed above.

The invention will now be illustrated in the experimental part givingproof of principle. The invention is further defined in the appendingclaims.

EXPERIMENTAL PART Example 1

Preparation of High Viscosity Agarose Solution

An agarose solution is prepared in a batch reactor by adding 40 g ofagarose to 1000 ml of distilled water under stirring at 95° C. After 2 hthe solution is cooled to 60° C. and 5 ml of aqueous NaOH (50%) and 1.5ml of 1,4-butane-dioldiglycidylether are added to the agarose solution.1,4-Butane-dioldiglycidylether will now intra-molecularly andinter-molecularly cross-link the polysaccharide chains and the viscositywill increase continuously. When the desired viscosity is reached thecross-linking reaction is stopped by neutralisation with 60% acetic acidto pH=7. Before the reaction started the viscosity was 180 centipoise(60° C., pH 7-8) and after 3.5 h of reaction the viscosity was 1200centipoise (60° C., pH 7-8) and after 5 h the viscosity was 2200centipoise (60° C., pH 7-8).

The viscosity at 3.5 h is 670% of the viscosity of the starting agarosesolution. At 5 h he viscosity is about 1200% of the starting agarosesolution.

Example 2

Solidifying the Agarose Solution to Beads (Gel) by Emulsification.

The emulsion media is made in an emulsion reactor by adding 45 g ofethyl cellulose (N-50 emulsifier) to 580 ml of toluene under stirring at60° C. The dissolving of N-50 in toluene takes approximately 2 h.

The stirring is regulated to 115 rpm. 400 ml of agarose solution (1200centipoise (60° C., pH 7-8)) from Example 1 is transferred to theemulsion media whereby drops of agarose solution are formed. After 0.5 hof emulsification the mixture is cooled during approximately 10 h tobelow 25° C. The beads were washed under stirring with ethanol 99.5%,which is decanted. Thereafter the beads were washed on a glass filterwith ethanol 99.5% and distilled water.

Cross-Linking the Gel

40 g Na₂SO₄ are added to a reactor containing a solution of 260 ml ofgel (drained) and 65 ml distilled water under stirring. The reactiontemperature is increased to 50° C. and 4 g of aqueous NaOH (50%) and 0.3g of NaBH₄ are added to the solution as well as 35 ml of aqueous NaOH(50%) and 33 ml of epichlorohydrin, which are added during a period of6-8 h. The reaction continues over night (ca. 16 h). The gel is washedwith distilled water and 60% acetic acid is added to obtain a pH=5-6.

Example 3

Solidifying the Agarose Solution to Beads (Gel) by Emulsification.

Analogous to Example 2. 60 g ethyl cellulose (N-50 emulsifier), 580mltoluene, and 400 ml agarose solution (2200 centipoise (60° C., pH 7-8))from Example 1

Cross-Linking the Gel

Analogous to Example 2. 30 g of Na₂SO₄, 200 ml of gel (drained), 50 mlof distilled water, 3 g of aqueous NaOH (50%), 0.2 g of NaBH₄, 27 ml ofaqueous NaOH (50%) and 26 ml of epichlorohydrin.

Example 4

Preliminary Determination of Kav for the Material Obtained in Examples2-3 and a Comparison with Sepharose 4 Fast Flow.

Sepharose 4 Fast Flow is commercially available from Amersham PharmaciaBiotech AB, Uppsala, Sweden). It is produced by a method comprisingsteps (a)-(c) above but utilising uncross-linked agarose (4%) in step(a). Included in the solution is also a cross-linker (epichlorohydrin).The results are given in the table. Dextran Example 2 Example 3Sepharose 4FF Mol. Weight Kav Kav Kav 21400 0.79 0.72 0.78 67000 0.620.55 0.62 196300 0.42 0.34 0.44 401300 0.19 0.16 0.29

Examples 2-3 utilise solutions containing 4% agarose, which isessentially the same as used for the manufacture of Sepharose 4 FastFlow. The results thus suggest that the use of a cross-linked butsoluble polysaccharide influences the porosity (Kav) of the final matrixto a very low degree, if any, for dextran of low and medium molecularweights. For proteins and other bio-organic compounds that are morecompact than dextran this means that Kav should be essentially constantup to at least 400,000 Dalton or more when increasing the viscosity ofthe agarose solution by cross-linking (solution I).

Example 5

Inclusion of Densifying Particles in Solution (a).

Preliminary results with densifying steel particles (Anval, 8.4 g/cm³,Anval, Torshälla, Sweden) strongly suggests that an increase inviscosity by including the cross-linked but soluble polysaccharide instep (a) will give a reduced amount of beads lacking densifyingparticles.

Having described the particular, desired embodiments of the inventionherein, it should be appreciated that modifications may be madetherethrough without departing from the contemplated scope of theinvention. The true scope of the invention is set forth in the claimsappended hereto.

1. In a method for the manufacture of a solid porous separation materialbased on a polysaccharide, including the steps of (a) providing anaqueous solution of a polysaccharide, and (b) solidifying the solutionby cooling to transform the solution to a gel, the improvementcomprising modifying the viscosity of the aqueous polysaccharide bycross-linking prior to step (b).
 2. The method of claim 1, furthercomprising a step of cross-linking of the polysaccharide gel of step(b), either simultaneously with, or subsequent to, step (b).
 3. Themethod of claim 1, wherein in step (b), the gel is solidified into theform of beads or a monolith.
 4. The method of claim 1, wherein thepolysaccharide used in step (a) is water-soluble above a certaintemperature and forms a gel at lower temperatures.
 5. The method ofclaim 4, wherein the polysaccharide is selected from the groupconsisting of dextran, agarose, cellulose, starch and pullulan.
 6. Themethod of claim 1, wherein, prior to step (b), the solution isdisintegrated to drops that are transformed to a gel in step (b).
 7. Themethod of claim 6, wherein the disintegration includes forming asuspension in which the polysaccharide solution is the discontinuousphase and an organic solvent the continuous phase.
 8. The method ofclaim 6, wherein the disintegration results in formation of free drops.9. The method of claim 8, wherein disintegration includes spraying thesolution through a nozzle or by atomizing the solution by a spinningdisc technique.
 10. The method of of claim 1, wherein densifyingparticles which have a density higher than the solution are dispersedinto the solution or coated with the solution before step (b).
 11. Themethod of claim 10, wherein the densifying particles have a density ≧1.5g/cm³ such as ≧3 g/cm³.
 12. The method of claim 10, wherein thedensifying particles constitute ≦95% (v/v) of the polysaccharidesolution.
 13. (canceled)
 14. The method of claim 1, wherein thepolysaccharide before step (a), or during or after the subsequentsequence of steps is functionalized with affinity groups.